Transcriptomic intra-tumor heterogeneity of colorectal cancer varies depending on tumor location within the colorectum

BackgroundIntra-tumor heterogeneity (ITH) of colorectal cancer (CRC) complicates molecular tumor classification, such as transcriptional subtyping. Differences in cellular states, biopsy cell composition, and tumor microenvironment may all lead to ITH. Here we analyze ITH at the transcriptomic and proteomic levels to ascertain whether subtype discordance between multiregional biopsies reflects relevant biological ITH or lack of classifier robustness. Further, we study the impact of tumor location on ITH.MethodsMultiregional biopsies from stage II and III CRC tumors were analyzed by RNA sequencing (41 biopsies, 14 tumors) and multiplex immune protein analysis (89 biopsies, 29 tumors). CRC subtyping were performed using consensus molecular subtypes (CMS), CRC intrinsic subtypes (CRIS), and TUMOR types. ITH-scores and network maps were defined to determine the origin of heterogeneity. A validation cohort was used with one biopsy per tumor (162 tumors).ResultsOverall, inter-tumor transcriptional variation exceeded ITH, and subtyping calls were frequently concordant between multiregional biopsies. Still, some tumors had high ITH and were classified discordantly. Proximal tumors showed ITH related to differences in the microenvironment, while ITH within distal tumors were cancer-cell related. This subtype discordancy reflected actual molecular ITH within the tumors. The relevance of the subtypes was reflected at protein level where several inflammation markers were significantly increased in immune related transcriptional subtypes, which was verified in an independent cohort (Wilcoxon rank sum test; p<0.05).ConclusionOur transcriptomic and proteomic analyses show that the tumor location along the colorectum influence the ITH of CRC, which again influence the concordance of subtyping.EGA study EGAS00001004668

背景 结直肠癌（colorectal cancer, CRC）的肿瘤内异质性（intra-tumor heterogeneity, ITH）会使包括转录学分型在内的分子肿瘤分类工作变得复杂。细胞状态差异、活检细胞组成差异以及肿瘤微环境均可能诱导肿瘤内异质性。本研究从转录组与蛋白质组层面解析肿瘤内异质性，旨在明确多区域活检样本间的分型不一致究竟是反映了肿瘤内部真实存在的生物学层面异质性，还是源于分类器稳健性不足。此外，本研究还探讨了肿瘤位置对肿瘤内异质性的影响。
方法 针对II期与III期结直肠癌肿瘤的多区域活检样本，本研究分别开展两项检测：通过RNA测序（RNA sequencing）分析41份活检样本（对应14例肿瘤），通过多重免疫蛋白分析检测89份活检样本（对应29例肿瘤）。采用共识分子亚型（consensus molecular subtypes, CMS）、结直肠癌固有亚型（CRC intrinsic subtypes, CRIS）以及肿瘤类型三种方案完成结直肠癌分型。本研究定义了ITH评分与网络图以解析异质性来源，并使用一个每例肿瘤仅获取1份活检样本的验证队列（共162例肿瘤）开展验证。
结果 整体而言，肿瘤间转录组变异程度高于肿瘤内异质性，且多区域活检样本间的分型结果通常具有较高一致性。不过仍有部分肿瘤呈现高肿瘤内异质性，导致分型结果不一致。近端结直肠癌的肿瘤内异质性与微环境差异相关，而远端结直肠癌的肿瘤内异质性则与肿瘤细胞自身特性相关。上述分型不一致现象实则反映了肿瘤内部真实存在的分子层面肿瘤内异质性。分型的相关性在蛋白质组层面得到印证：与免疫相关的转录组亚型中，多种炎症标志物水平显著升高，该结果在独立队列中通过Wilcoxon秩和检验得到验证（p<0.05）。
结论 本研究的转录组与蛋白质组分析表明，结直肠不同部位的肿瘤位置会影响结直肠癌的肿瘤内异质性，进而影响分型结果的一致性。本研究的EGA编号为EGAS00001004668。